Single-cell resolution analysis reveals the preparation for reprogramming the fate of stem cell niche in cotton lateral meristem

Abstract

Background: Somatic embryogenesis is a major process for plant regeneration. However, cell communication and the gene regulatory network responsible for cell reprogramming during somatic embryogenesis are still largely unclear. Recent advances in single-cell technologies enable us to explore the mechanism of plant regeneration at single-cell resolution.

Results: We generate a high-resolution single-cell transcriptomic landscape of hypocotyl tissue from the highly regenerable cotton genotype Jin668 and the recalcitrant TM-1. We identify nine putative cell clusters and 23 cluster-specific marker genes for both cultivars. We find that the primary vascular cell is the major cell type that undergoes cell fate transition in response to external stimulation. Further developmental trajectory and gene regulatory network analysis of these cell clusters reveals that a total of 41 hormone response-related genes, including LAX2, LAX1, and LOX3, exhibit different expression patterns in the primary xylem and cambium region of Jin668 and TM-1. We also identify novel genes, including CSEF, PIS1, AFB2, ATHB2, PLC2, and PLT3, that are involved in regeneration. We demonstrate that LAX2, LAX1 and LOX3 play important roles in callus proliferation and plant regeneration by CRISPR/Cas9 editing and overexpression assay.

Conclusions: This study provides novel insights on the role of the regulatory network in cell fate transition and reprogramming during plant regeneration driven by somatic embryogenesis.

Keywords: Cotton; Gene functional verification; Gene regulatory network; Plant regeneration; scRNA-seq.

Fig. 1

Primary vascular tissue of cotton hypocotyl is the primary target of Agrobacterium infection. a Agrobacterium-mediated genetic transformation and plant regeneration via somatic embryogenesis with cotton hypocotyls as explants, including callus induction, embryogenic callus induction, and plant regeneration via somatic embryogenesis. b The left picture shows etiolated cotton seedling under dark culture. Scale bar, 1 cm. The right picture shows the tissue types in the cross-section of cotton hypocotyl. Scale bar, 500 μm. c The comparison of three major types of stem cells in plants, including SAM, RAM, and cambium in vascular bundles. The vascular bundle of dicotyledons is cyclic annular and consists of xylem, phloem, and cambium. The vascular bundles of monocotyledons are scattered without cambium. d Specific expression of green fluorescent protein and far-red fluorescent protein driven by CaMV 35S promoter in primary vascular tissue (indicated by yellow arrow) after Jin668 and TM-1 hypocotyls infected with Agrobacterium for 48 h. Scale bar, 500 μm

Fig. 2

Single-cell RNA-seq and cluster annotation of cotton hypocotyl. a Overview of cotton hypocotyl scRNA-seq workflow. Protoplasts were isolated from hypocotyl sections of Jin668 and TM-1 with two independent biological repeats, respectively. 10 × Genomics platform was used for high-throughput sequencing. b, c UMAP visualization shows these cotton hypocotyl cells were grouped in to 9 clusters both in Jin668 and TM-1. Each dot indicates a single cell. Different colors indicate cell clusters. d Marker genes of each cell cluster in Jin668 and TM-1. e RNA in situ hybridization of 3 selected genes, including Ghir_A01G019320 in primary xylem (PX), Ghir_D11G036340 in xylem vessel precursors (XV), and Ghir_A10G018730 in phloem (indicated by red arrow). Scale bar, 50 μm

Fig. 3

Highly conserved cell-type clusters and heterogeneity genes between Jin668 and TM-1. a UMAP visualization of Jin668 and TM-1 clusters after alignment. The left is sample (Jin668 or TM-1), the right is cell type. b Venn diagram showing the number of shared and cultivar-specific expressed genes for each cell-type cluster of Jin668 and TM-1. c UMAP visualization of expression patterns of the genes related to SE, including auxin and wound response. The colors represent expression levels of these genes in individual cells. d Paraffin section showed vascular tissue proliferation of hypocotyl after induction. The red arrows represent the proliferation site

Fig. 4

Genes specifically expressed in primary vascular tissue cells of Jin668 and TM-1. a Differential expression gene (DEGs) analysis between Jin668 and TM-1 after induction at the same time. Colored by fold-change direction (padj ≤ 0.05 & log2FoldChange ≥ 1). b Gene expression trend analysis showed that the auxin influx gene LAX2, wound response gene WIND1, and ANAC071 had a reverse expression trend in Jin668 and TM-1. c, d Venn diagram showing the genes specifically expressed in the cambium (c) and primary xylem (d) of Jin668. The expression trend of auxin transport-related genes after induction at different times in cambium (c) and primary xylem (d)

Fig. 5

Pseudotime trajectory of primary vascular cells. a, b Pseudotime analysis of primary xylem, xylem vessel precursors, parenchyma, and cambium cells of Jin668 and TM-1. Each dot indicates a single cell, and the color of the upper right corner represents the starting point and end point of differentiation. c, d Expression patterns of representative auxin-related genes (WAT1 and IAA4) are shown over the course of pseudo-time. Color bar indicates the relative expression level. e Heatmap showing the expression of the branch-dependent genes over pseudo time. GO terms of SE-related are shown in the table on the right. The middle of the heatmap is the beginning of pseudo time. Both sides of the heatmap are the end of pseudo time. Color bar indicates the relative expression level. J(T)PX, primary xylem of Jin668 (TM-1); J(T)XV, xylem vessel precursors of Jin668 (TM-1); J(T)PC, parenchyma and cambium region of Jin668 (TM-1)

Fig. 6

RNA velocity field describes fate decisions of primary vascular cells in the hypocotyls of Jin668 and TM-1. a Aggregated the fate maps of cell clusters in Jin668 and TM-1 into primary vascular cells using directed edges. The pie charts to show cell fates averaged per cluster. Edges between clusters are given by transcriptomic similarity between the clusters. b–d The fate maps of directional aggregation and gene expression trend of local cell clusters. Fate maps, phase portraits, unspliced residuals, and smoothed gene expression trends are shown from left to right for these driver regulated genes

Fig. 7

Network analysis SE-related genes of Jin668 and TM-1 hypocotyl primary vascular cells. a, b Weighted correlation network analysis of scRNA-seq data of Jin668 (a) and TM-1 (b) reveals multiple modules of co-expressed genes of various sizes. The color bar beneath the dendrogram represents the module assignment of each gene. c–f Module visualization of network connections and associated function in Jin668 and TM-1. The reported SE-related intramodular hub genes are indicated by a red dot

Fig. 8

A proposed model of the molecular regulation of cotton plant regeneration via somatic embryogenesis. a Chromatin-modifying proteins repress or restrict expression of transcription factors (TFs) during SE. Red represents Jin668, blue represents TM-1, and gray represents monocotyledon. The symbols in the box represent the main differences between Jin668 and TM-1. It includes gene expression pattern, differential expressed genes, gene co-expression network, and pseudo-time analysis. b–e The main factors affecting SE and regulatory network of related genes. Including auxin (b), cytokinin (c), ethylene pathways (d), and wound induction process (e). The reported and newly identified regulatory genes under the single-cell resolution in this study based on gene differential expression, cell trajectory inference, RNA velocity fate determination, and co-expression gene interaction are labeled in the corresponding module. The red oval represents the gene specifically expressed in Jin668, and the gray arrow represents the predicted SE-related genes. Overall, Jin668 and TM-1 exhibited distinct regulation model including 20 core genes (such as LAX2, WOX4, AGL15, WIND1) during the plant regeneration process via somatic embryogenesis

Fig. 9

Phenotype of GhLAX1, GhLAX2, GhLOX3 knock out and overexpression callus with hypocotyls as explants. a Schematic view of gRNA1, gRNA2 target sites in the GhLAX1, and GhLOX3 and overexpression cassette of GhLAX2. b Paraffin sections of hypocotyls infected with Agrobacterium after induction on callus induction medium for 0, 24, and 72 h. The red box represents the proliferation site. c The phenotypes of different transgenic explants and control (P7N) at 20 days post-induction and the callus proliferation rate (CPR) of explants and control at 20 days post-induction. d The phenotype of callus on the GhLAX1 knock out and GhLAX2 overexpression explants at about 70 days post-induction. Scale bar, 100 μm. e Days of embryonic callus occurrence of different transgenic explants. f Morphology of somatic cell embryos of JOE1. Scale bar, 100 μm